microRNAs (miRNAs) encode ~22nt small RNAs that regulate deadenylation, translation, and decay of their target mRNAs. With the potential to regulate more that 30% of the human genes, miRNAs play fundamental roles in every aspect of biology from human development, to human disease including neuropsychiatric disorders and cancer. In animals, miRNAs are derived from characteristic hairpins processed by two sequential RNAse III enzymes, Drosha and Dicer. Our recent work has identified a novel microRNA processing pathway independent of Dicer function that depends on the catalytic activity of Argonaute2 as the initial processing step and is required for red blood cell development. Yet, the rules that govern Dicer vs. Argonaute processing and the downstream components of this pathway remain largely unknown. This proposal combines, biochemistry, mass spectrometry genetics and high-throughput sequencing with the aim to understand the structural and sequence factors that determine entry in the Argonaute vs. the Dicer processing pathway (Aim 1), identify the machinery downstream of Argonaute2 required to generate the mature miRNA through trimming and uridylation of the argonaute cleaved intermediate (Aim 2) and identify the processing requirements for all microRNAs during vertebrate development (Aim 3) using zebrafish as a model system. Abnormalities in microRNA processing have been associated with developmental defects and human cancer. In particular, miR-451 a microRNA exclusively processed by Argonaute2, is associated with glioma formation and blood disorders in humans. Thus, the identification of the machinery required downstream of Argonaute2 processing will help us understand how the dysfunction of microRNA processing might cause human birth defects and contribute to disease. In summary, the proposed experiments challenge a classical view in the field that all microRNAs are processed by Dicer have the long term goal of i) providing in-depth characterization of the processing, sequence and genomic origin of small RNAs during vertebrate development, providing an entry point to understand their function in vivo, and ii) uncovering an evolutionarily conserved machinery required to process small regulatory RNAs in vertebrates addressing fundamental questions in small RNA processing, gene regulation, RNA metabolism.